báo cáo khoa học: "Linkage disequilibrium in French of Drosophila melanogaster" pot

Original article Linkage disequilibrium in French of Drosophila melanogaster natural J Sanchez Prado L Charles-Palabost A Merỗot populations M Katz Universidad de Oviedo, Departamento de Genetica, Asturias, Spain Université F Rabelais, IBEAS, avenue Monge, Parc Grandmont, 37200 Tours, France Université de Paris v7/, Laboratoire de Génétique Quantitative et Mol6culaire, and Université de Paris VII, Laboratoire de Génétique des Populations, Tour 42-32, place Jussieu, 75005 Paris, France (received 23-9-1987, accepted 25-4-1988) Summary — Seventeen French natural populations of Drosophila melanogaster were analyzed to detect linkage disequilibrium between pairs of polymorphic allozyme loci The estimates of linkage disequilibrium were made from azygotic frequencies using both Burrows’ and Hills’s methods No difference between these methods was found The amount of significant linkage disequilibrium detected was small and similar to those in other natural populations of D melanogaster Out of the 15 combinations examined, only pairs, Adh-a-Gpdh and Est-C-Est-6, showed a consistent significant linkage disequilibrium in the populations studied However, for the first pair, the result was probably due to an association between the loci and the inversion (2 L) t of the second chromosome For the Est-C-Est-6 pair, the disequilibrium detected might result from an interaction effect between the genes inter se These results again show the difficulties in detecting linkage disequilibrium due to epistasis between allozyme genes in natural populations Drosophila melanogaster- linkage disequilibrium - enzymatic loci - French natural popula- tions Dộsộquilibre de liaison dans des populations naturelles franỗaises de Drosophila melanogaster Une analyse du déséquilibre de liaison a été effectuée pour locus enzymatiques dans 17 populations naturelles de Drosophila melanogaster Les estimations de ce déséquilibre ont été faites, partir des fréquences zygotiques, en utilisant les méthodes de Burrows et de Hill Aucune différence n’a été observée entre ces deux méthodes La quantité de déséquilibre décelée est faible et comparable celle trouvée dans d’autres populations naturelles de D melanogaster Sur les 15 combinaisons examinées, seules les associations Adh-a-Gpdh d’une part, Est-C-Est-6 d’autre part, montrent un déséquilibre significatif dans les populations étudiées Le déséquilibre Adhu a-Gpdh est probablement dû la liaison entre les gènes correspondants et l’inversion (2 L) t du second chromosome Au contraire, le déséquilibre Est-C-Est-6 pourrait être la conséquence d’interactions entre les gènes eux-mêmes Ces résultats soulignent nouveau les difficultés rencontrées dans la mise en évidence d’un déséquilibre de liaison véritablement dû une épistasie entre locus enzymétiques Résumé — Drosophila melanogaster - déséquilibre de liaison - locus enrymatiques - populations natu- Introduction Population studies of genetic variation are classically discussed in terms of single-locus variability measures, such as heterozygosities and changes in gene frequencies However, there is much interest in knowing the genetic structure of populations at the multilocus level The application of electrophoretic techniques to analyze genetic variation (Harris, 1966; Hubby and Lewontin, 1966) provides much information at the multilocus level, because a large number of genetic markers can be studied simultaneously in a single individual Therefore, investigations made on allozyme polymorphism involve the estimation of linkage disequilibrium in natural and experimental populations of a variety of organisms (see Hedrick et al., 1978, for a review) Various authors (e.g., Lewontin, 1974) have suggested that information about linkage disequilibrium among allozymes might be useful to explain the adaptive value of biochemical polymorphism But unfortunately, the results obtained by the authors studyng linkage disequilibrium at electrophoretically variable loci in natural populations of Drosophila melanogaster (Mukai and Voelker, 1977; Voelker et al., 1977; Langley et al., 1978; Inoue et aG, 1984; Yamazaki et al., 1984) are reconcilable with several models of population genetics Consequently, even in the absence of inversion, it is difficult to determine whether these results are due to epistatic natural selection or to random genetic drift However, we think that it is important to determine the nature and magnitude of linkage disequilibrium in natural populations, because the investigations may perhaps help in the study of interactions between genes and in developing new hypotheses about the mechanisms involved in the maintenance of allozyme polymorphism In this paper we report a study of linkage disequilibrium among polymorphic allozyme loci in 17 natural populations of D melanogaster collected from different regions of France Materials and Methods Collections Wild Drosophila melanogaster adults were collected and brought to the laboratory for electrophoresis All collections were made during the annual demographic burst of the species (between August and October) Populations studied populations studied are distributed from the North to the South of France (Fig 1); their origins listed below : (1) Venteuil near Epernay; (2) Verneuil near Epernay; (3) Vincennes near Paris; (4) Sbvres near Paris; (5) Ivry-sur-Seine near Paris; (6) Sainte-Genevi6ve-des-Bois near Paris; (7) Rann6e near Rennes; (8) Nevez near Quimper; (9) Chateaubriant; (10) M6n6tr6ol-sous-Sancerre near Sancerre; (11) Bonnac-la-C6te near Limoges; (12) Chessy-les-Mines near Villefranche-sur-Sa6ne; (13) Beynost near Montluel; (14) Le Curtelod near Yenne; (15) Montauban; (i 6) Tautavel near Perpignan; (17) Port-Vendres Only populations (1) and (2) were captured in wine-cellars; the others originated from fruits of the localities studied Two collections were made for populations (6) and (9), the first in 1983 and the second in 1984 Populations (1)-(5) and (17) were collected in 1984 and the others in 1983 The are Electrophoresis Electrophoresis was performed in horizontal starch gel with Poulik’s discontinuous buffer system Six polymorphic enzyme loci were studied, according to the techniques described by Charles-Palabost (1986) : acid phosphatase (Acph; 3:101.4), alcohol dehydrogenase (Adh; 2:50.1), esterase-C (Est-C; 3:47.6), esterase-6 (Est-6; 3:36.8), a-glycerophosphate dehydrogenase (a-Gpdh; 2:20.5), phosphoglucomutase (Pgm; 3:43.4) and Estimation of linkage disequilibrium In this study almost all the data were analyzed by a 2-allele system If more than alleles exist at a classes: the most frequent allele corresponding to the first class, locus, they have been grouped in and the others to the second Let us consider loci A and B, each having, respectively, alleles A-a (frequency of A : p) and B b (frequency of B: q), gametes are possible : AB, Ab, aB, and ab If the gametic frequencies are, respectively, f11, !2 f , 2l and , 22 f the linkage disequilibrium D is given by : In order to make the values of the parameter D less sensitive to change in gene frequency, several other measures of gametic disequilibrium are useful in various contexts The correlation coefficient R D/Vpg (1-p) (1-q) was used by Hill and Robertson (1968) and by Franklin and Lewontin (1970) However, in a sample of individuals taken from a population, the degree of linkage disequilibrium cannot be estimated directly from the genotypic frequencies when the coupling and repulsion heterozygotes cannot be distinguished In this case, estimation of linkage disequilibrium can be done in several ways Hill (1974) provides a maximum-likelihood method where the population is assumed to be random mating and in Hardy-Weinberg equilibrium at each locus In the case of codominant alleles per locus, the frequency of one gamete (for example AB) estimated by the maximum-likelihood method (f is given by a cubic equation : ) 11 with , , , II 12 21 N N N N!, and N corresponding, respectively, to the observed numbers of AABB, AABb, AaBB, AaBb, and total individuals in the sample Eq (1) the only unknown is f11 Hill suggests that an initial value : f11(4N + 2N + 2N + » l2 21 N! )l2N pq can be substituted into the right-hand side of (1) and the resulting expression regarded as an improved estimate and itself substituted into the right-hand side of (1 The iterative process is In continued until stability is reached and D obtained as : D = - pq A test for D is given by : K 11 f / D Npq (1-p) (1-q), with Kfollowing the chi-square distribution with one degree of freedom A second approach, suggested by Burrows (see Cockerham and Weir, 1977 and Langley et al., 1978), is simply used to estimate the overall covariance of non-allelic genes in individuals This method does not require that one distinguish between the types of double heterozygotes and know the mating system Burrows’s parameter is estimated by : ! 1/2 (4N + 2N + 2N /N 1N /N 11 121 21 + 2N 2pq A test for A is given by : X NA (1-p) (1 !) where X is approximately a X /M 22 2 /pq 2 distribution with one degree of freedom (Cockerham and Weir, 1977) The correlation coefficient based on Burrows’s estimation is : R A/2 ! pq (1-p) (1-q) In any population, all the loci are not necessarily in Hardy-Weinberg equilibrium Therefore, we used not only Hill’s method, which assumes that the loci are in accordance with the Hardy-Weinberg law, but also Burrows’s estimation Moreover, it was interesting to compare the results = = = = = = obtained by both methods because this was done only in few cases Results TableI gives, for each population, the number of flies analyzed per locus and the frequencies of the most common allele at each locus With regard to the distribution of allelic frequencies, the populations collected in 1983 were analyzed in another paper (Charles-Palabost et al., 1985), and those of 1984 will be analyzed later Concerning the goodness of fit to Hardy-Weinberg equilibrium, the use of the X test is not appropriate in some cases, since the expected numbers of genotypes are too small Therefore, each a value given in TableI is the probability that the genotypic frequencies distribution of a random sample are farther from the expected Hardy-Weinberg model than the corres- ponding observed distribution These values obtained by means of Monte-Carlo the real frequencies and under the null hypothesis in which the populations are in Hardy-Weinberg equilibrium This test is consequently frequency independent We observe that 21 a values out of 101 are significant and among these 21 significant values, 10 are due to the presence of a rare genotype in the samples It means that generally, the observed frequencies of heterozygotes were simulations, using the observed allelic frequencies as per locus in each population are in good Hardy-Weinberg law A significant excess Gpdh locus of the S6vres population agreement with those expected under the of heterozygotes was found only at the a- Table II shows the frequencies of the observed heterozygotes for each locus and population Classically, the amount of variation differs greatly from one locus to another The average heterozygosity over the loci analyzed ranges from 0.092 in the Nevez population, to 0.250 in the Ivry-sur-Seine and S6vres populations Except for Nevez, the mean heterozygosities obtained are similar to those estimated previously in other French natural populations of D melanogaster (Girard and Palabost, 1976) The values of linkage disequilibrium estimated by Burrows’ (A and R and Hill’s ) b methods (D and R are given in Table III for the unlinked loci (located on different chro) h mosomes) and in Table IV for those linked (located on the same chromosome) The use of the x distribution in order to determine the significance level of a linkage disequili2 brium implies that in a sample of 100 individuals, the frequencies of the most common alleles at each of the loci must be smaller than 0.85 (Montchamp-Moreau, 1985) Thus, the significance levels in Tables III and IV correspond to the probability that the linkage disequilibrium estimated from a random sample is greater than the linkage disequilibrium estimated from the sample analyzed These probabilities were obtained using MonteCarlo simulations, under the null hypothesis of a disequilibrium equal to This test is independent of the distribution, but assumes that the observed allelic frequencies are the real frequencies in the populations We can note that the values of D and A are very similar for unlinked as for linked loci By contrast, the correlation coefficients R (Hill’s estimah tion) and R (Burrows’s estimation) are different and, in most cases, R is smaller in b b absolute values than R (161 cases out 216 values) When R R (in 55 cases), no h b h double heterozygotes are present in the samples and 2D; this result is particularly evident for unlinked loci With Hill’s method, 23 out of the 216 comparisons made between pairs of loci are significant, which represents a percentage of 10.6 The percentages obtained, respectively, for the unlinked and linked loci are 10.5 (13/124) and 10.9 (10/92) With Burrows’s method, these values are 15.3% (33/216) for all the loci, 11.3% (14/124) and 20.6% (19/92), respectively, for unlinked and linked loci In the present study, out of the 15 combinations between allozyme loci, only the pair Est-C-Est-6 shows a significant linkage disequilibrium in most of the populations : D values out of 18 populations sampled (22%) and values (44%) are significant (Table IV) Using combined data of all the populations, a significant deviation was obtained only in cases : for the Est-C-Est-6 pair and also for Adh-a-Gpdh With Hill’s estimation, the values are, respectively, for Adh-a-Gpdh and Est-C-Est-6 pairs : D 0.0116 (P < 0.0097 (P < 0.01), R 0.0943 The corresponding 0.0991, and D 0.01), R h h = = = = - = - values with Burrows’s estimation 0.0132 (P0.01), Rb=-0.0643 = - =&dquo;0.0548,andA= b are :A=-0.0129(P